Calcium-Responsive Transcriptional Activation

HEK-CaSR cells were seeded in 48-well plates and transiently transfected with 100 ng/ml Gα₁₁ WT or mutant proteins, 100 ng pGL4-SRE luciferase reporter construct, and 10 ng/ml pRL control vector for 48 hours (Promega). Cells were incubated in serum-free media for 12 hours, followed by treatment of cells for 4 hours with 0–10 mM CaCl₂. Cells were lysed and assays performed using Dual-Glo luciferase (Promega) on a Veritas Luminometer (Promega), as previously described (25 (link), 45 (link)).

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Gorvin C.M., Hannan F.M., Howles S.A., Babinsky V.N., Piret S.E., Rogers A., Freidin A.J., Stewart M., Paudyal A., Hough T.A., Nesbit M.A., Wells S., Vincent T.L., Brown S.D., Cox R.D, & Thakker R.V. (2017). Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy. JCI Insight, 2(3), e91103.

Publication 2017

Dual-Glo luciferase Veritas Luminometer

Assays Cells Luciferase Mutant proteins Pgl4 Promega Serum free media

Corresponding Organization : University of Oxford

Other organizations : University of Liverpool, Mary Lyon Centre at MRC Harwell, Medical Research Council, University of Ulster

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Protocol cited in 3 other protocols

Variable analysis

independent variables

Gα11 WT or mutant proteins (100 ng/ml)

dependent variables

SRE luciferase reporter activity

control variables

PGL4-SRE luciferase reporter construct (100 ng)
PRL control vector (10 ng/ml)
Serum-free media incubation for 12 hours
CaCl2 treatment (0-10 mM) for 4 hours

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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